Candidatus Liberibacter is a filamentous bacterium that lodges in the phloem sieve tubes, and may have round forms when its cell cycle ends. It is transmitted by insect vectors, where it is present in the hemolymph and salivary glands (Jagoueix, Bové, & Garnier, 1994). This bacterium affects different crops belonging to the Rutaceae, Solanaceae and Apiaceae families (Liefting, Pérez-Egusquiza, Clover, & Anderson, 2008; Aguilar, Segonda, Bextine, McCue, & Munyaneza, 2013). A form of dispersion of Ca. Liberibacter is through infected seed, which is part of the primary inoculum in the field and influences the initial incidence of the disease and the behavior of the epidemic in the field (Camacho- Tapia et al., 2011).
Phylogenetic studies of the 16S rRNA region indicate that Ca. Liberibacter belongs to the α-proteobacteria, and that it is related to bacterial genera of the α-2 proteobacteria subdivision, such as: Bartonela, Bradyrhyzobium, Agrobacterium, Brucella and Alfipia (Jagoueix et al., 1994). Observations with transmission electron microscopy show that the bacterium is 0.2 to 0.3 μm in diameter, has a characteristic membrane of gram-negative bacteria, has a peptidoglycan layer, which is barely visible, and shows no evidence of flagella or pili (Jagoueix et al., 1994).
Isolating Ca. Liberibacter in artificial culture medium is difficult, making its biochemical characterization impossible, and so its genus classification as Liberibacter, from Liber (bark) and bacter (bacteria), remains only as a proposal. Based on comparing nucleotide sequences of ribosomal protein genes, three species found in Rutaceae, namely Ca. Liberibacter africanus, Ca. Liberibacter asiaticus and Ca. Liberibacter americanus, and a fourth species, Ca. Liberibacter solanacearum, detected in Solanaceae, have been proposed (Jagoueix et al., 1994; Liefting et al., 2008).
The aim of this review was to provide an overview of the biological, ecological, epidemiological and management aspects of Candidatus Liberibacter, which is associated with diseases such as citrus Huanglongbing, Zebra chip and chili pepper variegation.
Isolation of Candidatus Liberibacter
The first attempts to isolate Liberibacter were through co-cultures with actinobacteria closely related to Propionibacterium acnes; in these, the colonies appeared after a long time and it was difficult to keep them free to transfer them consecutively to a fresh culture medium (Davis, Mondal, Chen, Rogers, & Brlansky, 2008). In a second study, another medium designated Liber was designed and evaluated, obtaining irregular growth and bacterial colonies ranging from 0.1 to 0.3 mm after three to four days at 28 °C; subsequently the colonies did not increase in size, and their viability was lost after four to five reisolations. Therefore, the difficulty of isolating it precludes testing for its biochemical study (Sechler et al., 2009).
Parker et al. (2014) indicated that adding grapefruit (Citrus paradisi Macfad) juice to the culture medium prolongs viability of Ca. L. asiaticus; on the other hand, it decreases in sinusoidal fashion in conditions such as lower pH, presence of sugars and abundance of minerals and amino acids (Parker et al., 2014).
Although Ca. L. americanus has an intact outer membrane, most of the genes required for lipopolysaccharide (LPS) biosynthesis, which are found in Ca. L. asiaticus and Ca. L. solanacearum, are absent, together with lpxA, lpxB and lpxC, which are involved in the early steps of lipid biosynthesis. The lack of LPS in gram-negative bacteria can be compensated by the production of other lipid bases in the outer membrane (Lin & Gudmestad, 2013).
Ca. L. americanus and Ca. L. solanacearum appear to have a greater metabolic capacity for some amino acids and vitamins. Ca. L. solanacearum has the complete pathway for the production of arginine, glutamine and ornithine (arginine and glutamine from ornithine). Genes for the synthesis of glutamine from glutamate are present in these bacteria (Wulff et al., 2014). The symptoms of generic yellows and decline, often readily confused with nutritional deficiencies, are due to starch accumulation, indicating a disruption of phloem transport (Nelson, Munyaneza, McCue, & Bové, 2013).
In the Ca. L. asiaticus genome, two bacteriophages called SC1 and SC2, which belong to the family Podoviridae, corresponding to the order Caudovirales, were found (Zhang et al., 2011a). The former seems to be activated when the bacteria infects citrus. The latter, apparently, is a degenerated form of SC1 lacking lysis genes, but carries most of those that could be important in lysogenic conversion; that is, those that are expressed in the bacteria and alter their physiology, increasing their virulence and pathogenicity, as well as causing the appearance of new haplotypes and forms of the disease. SC2 is replicated as an excision plasmid when its Ca. L. asiaticus host is infecting either plants or psyllids.
Studies indicate that Bactericera cockerelli, in addition to carrying Ca. L. solanacearum, has three prokaryotic intracellular bacteria (symbionts) associated with it: the primary or obligate symbiont (Carsonella rudii) (Thao et al., 2000) and two facultative ones, a secondary (S) and two different strains of Wolbachia (Nachappa, Levy, Pierson, & Tamborindeguy, 2011). These symbionts are heritable and transmitted from mother to offspring (Nachappa, Shapiro, & Tamborindeguy, 2012).
The primary symbiont affects the fundamental biological process of the insect with the host, and is required for the survival and development of the psyllid, in addition to the facultative symbionts that influence it ecologically. For example, Wolbachia spp. can manipulate reproduction in the insect, providing infected females with a reproductive advantage compared to uninfected females. This increases the dispersion of Wolbachia spp., in addition to encouraging insect growth and increasing the percentage of vectors for transmitting Liberibacter in Solanaceae (Nachappa, Levy, Pierson, & Tamborindeguy, 2014).
Candidatus Liberibacter in different hosts
Huanglongbing (HLB), also known as citrus greening, is a disease in citruses associated with the infection of three species of α-proteobacteria: Ca. Liberibacter asiaticus, Ca. Liberibacter americanus and Ca. Liberibacter africanus (Bové, 2006). HLB symptoms include blotchy mottling and chlorosis, which can be confused with nutritional deficiencies, as well as stunting and reduced foliage as the disease progresses, and in extreme cases the trees die from the infection (Gottwald, da Graça, & Bassanezi, 2007).
The yield of affected trees is reduced, and they may produce small, poor-quality fruit. Commercial varieties are the most susceptible (Wang et al., 2009). Ca. L. asiaticus and Ca. L. americanus are transmitted by Diaphorina citri (Kuwayama) (Capoor, Rao, & Visnawanath, 1967; Yamamoto et al., 2006), whereas Ca. L. africanus is transmitted by Trioza erytrae (Del Guercio) (McClean & Oberholzer, 1965). In the United States, Ca. L. asiaticus was identified in Florida in 2005 (Halbert, 2005).
On the other hand, there is evidence that the disease known as Zebra chip, found in potato (Solanum tuberosum), originated in North America and later spread to other producing countries. The pathogen associated with this disease is Ca. Liberibacter solanacearum, which causes economic losses to the potato industry in the United States. Zebra chip was detected in Saltillo, Mexico, in the 1990s, and was first associated with a phytoplasma (Secor & Rivera-Varas, 2004). In early 2000 it was reported in commercial potato fields in Pearsall and Rio Grande Valley, Texas. By 2006, the disease had spread to other producing states, such as Arizona, Kansas, Colorado, California, Nevada and New Mexico (Munyaneza, Goolsby, Crosslin, & Upton, 2007b; Liefting et al., 2008).
In 2008, in Auckland, New Zealand, Zebra chip symptoms detected included: chlorotic apices, general mottling of the leaves, curling of the midveins, stunting of the plants, reduced yield, flecking and streaking in the raw tubers that became marked when fried, and in some cases tuber deformation (Munyaneza, Crosslin, & Upton, 2007a; Liefting et al., 2008).
Candidatus Liberibacter solanacearum was detected in the carrot psyllid (Trioza apicalis) in carrot plantations in Finland infected by this insect. Trioza apicalis is a serious insect pest in northern and central Europe, where it can cause up to 100 % crop loss. In 2009, in southern Sinaloa, Mexico, this bacterium was found as pathogen in the pepper crop; plant symptoms resembled those of yellow psilid that occurs in potato, but they also showed shortened internodes and chlorotic apical growth (Munyaneza, Sengoda, Crosslin, Garzon-Tiznado, & Cárdenas-Valenzuela, 2009). For their part, Camacho-Tapia et al. (2011) demonstrated that this bacterium is responsible for chili pepper variegation observed in commercial plantations of this solanacous crop in the Yurécuaro region of Michoacan, Mexico, and that it can be transmitted by seed.
In 2013, in Honduras, Ca. L. solanacearum was reported in tobacco (Aguilar et al., 2013), and in 2014 it was found in pepper (Munyaneza & Segonda, 2014). New species of this bacterium have been documented, as in the case of Ca. Liberibacter europaeus which was detected in Italy as an endophyte in pear and its associated psyllid (Cacopsylla pyri) (Raddadi et al., 2010).
Temperature has a significant effect on the development of Liberibacter associated with HLB and the symptoms of the disease. Ca. L. africanus and Ca. L. americanus are reported as heat-sensitive, while Ca. L. asiaticus apparently tolerates it. The heat sensitivity of the pathogen may explain the geographical distribution of these species of Liberibacter and their insect vectors (Munyaneza, Sengoda, Buchman, & Fisher, 2012).
Candidatus L. africanus is influenced by heat, as the development of symptoms occurs from 30 to 32 °C. T. erytrae occurs in cool climates, and is also sensitive to high temperatures combined with low relative humidity. By contrast, Ca. L. asiaticus is heat-tolerant; its symptoms develop in conditions of low humidity and temperatures above 35 °C, which is the temperature that D. citri stands (Bové, 2006; Lopes et al., 2009). Esquivel-Chávez et al. (2012) indicate that Mexican lime is more susceptible to Ca. Liberibacter asiaticus than Persian lime (C. latifolia) and sweet orange (C. sinensis). Flores-Sánchez et al. (2015) mention that the organoleptic characteristics of Persian lime are affected in the presence of Ca. Liberibacter asiaticus, and production is focused on secondary-growth branches and the outside of the tree canopy (Esquivel-Chávez et al., 2012).
At 17 °C or less, Ca. L. solanacearum is affected, but this does not prevent the development of Liberibacter in the plant, whereas at 32 °C or higher, the bacterium and the symptoms caused by it are inhibited. Therefore, it is reported that optimum development of Ca. L. solanacearum occurs from 27 to 32 °C. Heat sensitivity may explain the incidence and severity in producing areas (Munyaneza et al., 2012).
The optimum development of the psyllid B. cockerelli is at 27 ° C, while oviposition, hatching, development and survival are delayed or reduced at 32 °C, and cease from 35 °C (List, 2009). A single generation can be completed in three to five weeks, depending on temperature conditions that are favorable to Ca. L. solanacearum and development of “Zebra Chip” symptoms. Therefore, it is believed that there is coevolution among the species of Liberibacter and its insect vector, for sensitivity to temperature and relative humidity (Munyaneza et al., 2012).
Candidatus Liberibacter is restricted to the phloem sieve elements of the host, and the salivary glands of the insect vector. In the phloem of plants infected by Ca. L. solanacearum, aggregations of phenolic components, peroxidases, oxidases, polyphenols, chitinases, free amino acids and sugars (sucrose, glucose and fructose) are found (Rashed, Wallis, Paetzold, Workneh, & Rush, 2013). These responses appear between the third and the fifth week after infection (Rashed et al., 2013).
Increased levels of reducing sugars and amino acids would contribute to increased browning when frying potatoes, because acrylamide formation occurs with the use of these components as substrates at frying temperature via the Maillard reaction (Wallis et al., 2014).
Samples collected in different citrus-growing regions with HLB problems reveal that there is anatomical deformation associated with the disease. Light microscopy shows multiple swellings located in the necrotic phloem and dispersed throughout the vascular system, plus massive accumulation of starch, aberrations in cambial activity and excessive formation of phloem (Schneider, 1967).
Phloem necrosis could cause blockage of the nutrient translocation stream, leading to anatomical changes such as yellow blotching and vein yellowing. One of the most notable changes in the phloem of infected tree leaves is the swelling of middle lamella between cell walls surrounding sieve tubes; also, highly symptomatic old leaves showed collapses of the phloem cells close to the xylem (Folimonova & Achor, 2010). A relationship of increased HLB incidence in citrus orchards when Phytophthora nicotianae, which affects the fibrous roots, is present, than when only Ca. L. asiaticus is there, has been reported (Ann, Ko, & Su, 2004; Graham, Irey, & Taylor, 2011).
Detection of Ca. L. asiaticus in citrus roots indicates that it is able to move and multiply in root tissue; moreover, it causes root damage before disruption of phloem in the leaves (Graham, Johnson, Gottwald, & Irey, 2013).
B. cockerelli, carrier of Ca. L. solanacearum, can inoculate healthy plants during feeding; in this, the pathogen multiplies and spreads systemically throughout the host. Knowledge of the precise inoculation mechanism and transmission efficiency of the pathogen by B. cockerelli can be used to make predictions of the epidemic and develop integrated management strategies for the disease (Rashed, Nash, Paetzold, Workneh, & Rush, 2012).
When Ca. L. solanacearum is distributed throughout the plant, the amount of bacteria acquired by B. cockerelli is not influenced by the insect feeding site; however, if Ca. L. solanacearum is not fully distributed, the insects tend to acquire high amounts of pathogen when they feed on different areas of the plant or stem (Rashed et al., 2012). The number of vector insects can affect the time it takes for symptom expression, due to difference in initial injected inoculum load; although after bacterial multiplication and systemic movement within the host, the disease increases at the same rate. The number of vectors is an important factor for the success of the inoculation; however, the success rate is independent of the amount of pathogen in B. cockerelli (Rashed et al., 2012).
Rashed, Workneh, Paetzold, Gray, and Rush (2014) indicated that in potato there is no physiological stage where it is more susceptible to Ca. L. solanacearum. Incidence is related to the movement of the insect vector, and severity of symptoms is independent of the bacterial titer.
Epidemiology of Candidatus Liberibacter
Epidemiological studies show that Ca. Liberibacter species have an adjustment to β-binomial distribution, indicating the presence of aggregations of infected plants, except for cases where the incidence is less than 0.20 %, since the trend would indicate random distribution. Such aggregations are due to the different reinfection cycles and the distribution of the insect vector (Madden & Hudges, 1995; Gottwald et al., 2007; Henne, Workneh, & Rush, 2012).
Insect populations, carriers of Ca. L. solanacearum, tend to decrease by the amount of bacteria located in their gastrointestinal tract. It is also reported that there is a negative correlation between the nymph survival percentage and this pathogen, which affects the reinfection cycles; B. cockerelli adults have greater transmission efficiency compared to the nymphal stages (Nachappa et al., 2014).
Management of Candidatus Liberibacter
Measures to control Zebra chip depend on early detection and control of the insect vector (Ravindran, Levy, Pierson, & Gross, 2011). Due to the absence of genetic resistance, insecticides are applied to reduce the presence of the insect vector, for example thiamethoxam and abamectin (Gottwald et al., 2007; Lin & Gudmestad, 2013). Hoffman et al. (2013) indicated that thermotherapy can control HLB. The first reports of these practices were in China, where Ca. L. asiaticus was eliminated in budwood and seedlings after the material was exposed to hot air of 49 to 50 °C for 50-60 min (Lo, Lo, & Tang, 1981; Lo, 1983). Immersing citrus seedlings infected by Ca. L. asiaticus in hot water (47-50 °C) for 6 min causes heat stress and can induce defense responses in plants that help them fight the infection (Hoffman et al., 2013).
Ca. Liberibacter control focuses on reducing inoculum sources, such as the use of disease-free plants, removal of symptomatic trees and chemical control of the insect to reduce the transmission of the bacterium. In the seventies the use of tetracycline by direct injections into the trunk of HLB-affected citrus trees was assessed in South Africa, China and Indonesia, which resulted in a reduction of symptoms in the treated species. This practice was discontinued because tetracycline only has a bacteriostatic effect, so applications are required each year; also, after many injections, phytotoxicity is manifested in treated trees (Lo et al., 1981).
The combination of penicillin G and streptomycin has an antimicrobial effect against Ca. L. asiaticus. Penicillin G potassium is quickly assimilated by plants and is relatively less phytotoxic. The main mechanism of streptomycin action is binding to the bacterial 30S ribosome; it changes its shape and inhibits protein synthesis by causing the misreading of mRNA. According to Zhang et al. (2011b), this same treatment may also be feasible for the control of Ca. L. solanacearum.
Genetic transformation has been proposed as a tool to induce HLB resistance. In other crops, such as spinach.
antimicrobial peptides, which serve to control gram-negative and gram-positive bacteria, have been used. Iftikhar, Rauf, Shahzad, and Zahid (2014) transformed citrus species with antimicrobial genes (LIMA and ATTE) and grafted them onto budwood infected with Ca. L. asiaticus; subsequently, when analyzed by PCR, the bacterium was not found in the rootstock. Joubert and Stassen (2000) found that selective pruning by removing damaged branches significantly reduces the incidence of the disease; moreover, there is a positive impact on fruit size and yield.
The Liberibacter are important globally. According to the review, it has been found that the bacterium affects both plant yield and quality (Wang et al., 2009). The vectors of this bacterium are of great importance. Ca. L. asiaticus and Ca. L. americanus are transmitted by Diaphorina citri (Kuwayama) (Capoor et al., 1967; Yamamoto et al., 2006), and Ca. L. africanus by Trioza erytrae (Del Guercio) (McClean & Oberholzer, 1965). For Solanaceae, the main vector is Bactericera cockerelli. Therefore, most of the studies are related to the interaction of the pathogen with its vector. It should be kept in mind that due to the translocation of Liberibacter, some authors indicate that when the bacterium is distributed throughout the plant, the site where the insect vector feeds does not matter; however, when Liberibacter is not dispersed, high densities thereof occur in stem and petiole (Rashed et al., 2012). Even so, when there is contagion by the insects, their density may affect the time it takes for disease expression, due to difference in initial injected inoculum load (Rashed et al., 2012).
The system becomes complex in the case of B. cockerelli; it has been found that in addition to carrying Ca. L. solanacearum, the insect has three prokaryotic intracellular bacteria (symbionts) associated with it. These symbionts are heritable and transmitted from mother to offspring (Nachappa et al., 2011).
Ca. Liberibacter, by competing with the B. cockerelli symbionts, causes these populations to decrease due to the amount of bacteria located in the gastrointestinal tract, thereby affecting the insect’s physiological states. Adults have higher transmission efficiency compared to the nymphal stages of B. cockerelli because the survival rate is lower in nymphs (Nachappa et al., 2014).
One of the control mechanisms for these bacteria is to reduce the inoculum sources, such as by using disease-free plants, removing symptomatic trees and applying chemicals to control the insect vector to reduce the transmission of the bacterium. In addition, pathosystems as an integrative system need to be further studied using epidemiological tools, in order to have a better understanding of the interaction in the field and establish control measures.
Candidatus Liberibacter is of great economic importance. Knowing its biological, ecological and epidemiological aspects provides a better understanding of its relationship with the pathosystem where it is found. Understanding the pathosystem provides the tools to design control strategies and restore balance to the agricultural system.
The insect vectors of Ca. Liberibacter have co-evolved depending on the optimum climate conditions for their development. Moreover, these bacteria evolve and may have genetic variations, such as those identified in the haplotypes of Ca. L. solanacearum where severity is influenced by these variations. Changes in haplotypes have an impact on the incidence and severity of the bacterium, affecting the speed of the onset of symptoms and the formation of aggregations of diseased plants.
Insect vector control is necessary, since it is the most common form of dissemination in the various species of Liberibacter; although seed transmission also has an impact, the introduction of the pathogen from the seedling stage creates a source of primary inoculum, which affects the formation of the foci of infection and the incidence of diseased plants in the field.